Microfabricated aperture-based sensor

ABSTRACT

A chemical sensor includes an enzyme layer, a diffusion layer and an analyte barrier layer positioned over the diffusion layer. Apertures are formed by microfabrication in the analyte barrier layer to allow controlled analyte flux to the diffusion layer.

TECHNICAL FIELD

[0001] This invention relates to the detection of molecules (analytes)present in fluids such as blood. More particularly, the inventionrelates to the detection of organic molecules in vitro using anamperometric catalytic-based sensor. In specific embodiments of theinvention, sensors formed by microfabrication processes and having noveldevice designs can be used to perform assays of various molecules,including glucose, lactate, cholesterol, pyruvate, sarcosine, bilirubin,and creatinine, present in blood and other bodily fluids.

BACKGROUND OF THE INVENTION

[0002] Assaying bodily fluids such as blood for levels of variousorganic molecules is useful in the treatment of diseased states. Forexample, diabetes mellitus is a disease characterized by poor regulationof blood glucose levels. The traditional treatments for mild forms ofthis disease, including adult onset diabetes, have included diet andexercise. More severe forms, however, require administration of insulin.One of the drawbacks of administering insulin is the possibility ofinsulin shock, caused by rapid decrease in blood glucose levels (glucoseimbalance) due to unintended over medication. Insulin shock is, however,only the most severe manifestation of glucose imbalance. Theconsequences of chronic glucose imbalance (both over and undermedication) are well documented and include damage to blood vessels andvarious body organs. Blindness is common, as is the loss of circulationin the extremities.

[0003] Accurate measurement of blood glucose levels would enable thepatient to modulate insulin dosage and avoid the effects of chronicglucose imbalance. One example of a prior art attempt at glucosemeasurement is a glucose sensor as disclosed in U.S. Pat. No. 3,542,662.In this device, an enzyme-containing membrane is disposed between afluid being assayed and a first oxygen sensor electrode. A similarmembrane not containing enzyme is disposed between the fluid and asecond reference oxygen sensor electrode. A certain portion of theoxygen diffusing through the enzyme-containing membrane is consumed byequimolar reaction with glucose catalyzed by the enzyme and is thereforeunavailable for detection by the first oxygen sensor electrode. Thesecond, reference oxygen sensor electrode, in which the membrane doesnot include enzyme, determines the concentration of oxygen that wouldhave been detected had not the enzyme-promoted reaction occurred. Thedifference in oxygen detected by the two electrodes is indicative of theglucose concentration.

[0004] A problem with this device is that the levels of oxygen andglucose in the blood are less than stoichiometric. In particular, theamount of oxygen is less than that needed to convert all the glucose.Thus the sensor can become oxygen limited and not respond accurately athigh glucose concentrations.

[0005] In order to bring the levels of glucose and oxygen tostoichiometric balance and thus create a device that gives accurateresults over the complete range of glucose concentrations found inblood, it has been proposed to design sensors that reduce the amount ofglucose reaching the enzyme layer relative to oxygen. This could beaccomplished in theory by providing a membrane layer which issignificantly more permeable to oxygen than glucose. U.S. Pat. No.4,650,547, described more fully hereinbelow, provides a generaldescription of this concept.

[0006] Implementing this approach has heretofore been difficult,however, due to the prior art's inability to precisely and reproduciblycontrol the permeability of the membrane. Without such precise control,the glucose flux reaching the enzyme layer may not be sufficientlyattenuated. Problems can also arise due to the presence of interferantmolecules, e.g. ascorbate and urate. The determination of creatininelevels, which is used to measure renal function, is an example of ananalyte that requires removal of these interferants.

[0007] U.S. Pat. No. 4,933,048 relates to water permeable, ionimpermeable membranes microfabricated over a hydrogel layer leaving anopening for ion exchange. Figure 2 of the '048 patent illustrates astructure where the opening is formed by having the hydrogel layerextend beyond the ion impermeable layer. Alternatively, the ionimpermeable layer can cover the entire hydrogel layer with holes formedbeyond the perimeter of the underlying electrode (column 7, line 1).Holes can be formed by laser perforation or other methods. The apertureis formed at a distance from the electrode and the function of the smallopening is to provide a low impedance electrolytic junction.

[0008] Glucose sensors using non-microfabricated or “macro” electrodesare known. See, for example, Fischer, U. and Abel, P., Transactions ofthe American Society of Artificial Internal Organs 1982, 28, 245-248(Fischer et al.); Rehwald, W., Pflugers Archiv 1984, 400, 348-402; U.S.Pat. Nos. 4,484,987; 4,515,584; and 4,679,562; and UK Patent Application2,194,843. However, no aspect of thin-film processing is described inthese documents.

[0009] Fischer et al. discloses a non-microfabricated glucose sensorwith a Teflon® membrane which is mechanically perforated. Glucose canonly enter through the perforation whereas oxygen can pass through theTeflon®, thus adjusting the stoichiometry in the enzyme layer andlinearizing the response. There is no teaching of optimizing orcontrolling the dimensions of the perforation. The Fischer et al.document is also silent on the use of microfabrication. East Germanpatent DD 282527 appears to correspond to this publication but does notname Fischer as an inventor.

[0010] U.S. Pat. No. 4,484,987 relates to a linearized glucose sensorbased on the concept of providing a layer with hydrophobic regions in ahydrophilic matrix where glucose can permeate the latter but not theformer, and oxygen can permeate both regions (see description of FIG. 1thereof). In an alternative embodiment, shown in FIG. 4, a hydrophobiclayer includes spaced small openings through which glucose molecules canpass. However, the '987 patent provides no teaching of how thedimensions or location of the openings are controlled and is silent onmicrofabrication.

[0011] U.S. Pat. No. 4,650,547 discloses a glucose sensor where ahydrophobic gas permeable membrane is placed over a hydrophilicenzyme-containing layer, where only the perimeter or peripheral edgethickness surface of the hydrophilic layer is exposed to the sample(FIG. 5). Glucose can only enter the hydrophilic layer at the perimeterand diffuse parallel to the plane of the layer, whereas oxygen can besupplied across the entire surface of the hydrophobic layer (column 6,line 3).

[0012] Anal Chem 57, 2351, 1985 provides teaching for making a relatedcylindrical device where the gap between a platinum wire electrode and agas permeable cylindrical coating is filled with an enzyme gel. There isno teaching, however, of microfabrication. U.S. Pat. No. 4,890,620relates to a similar structure and method based on a differentialmeasurement with a pair of sensors. An implantable version is disclosedin U.S. Pat. No. 4,703,756.

[0013] Regarding lactate and creatinine, there is comparatively littlesensor literature. In Clin. Chem. 29, 51, 1983, there is proposed anamperometric creatinine sensor using three enzymes coupled to theproduction of hydrogen peroxide. This document also includes adifferential measurement where one sensor measures creatine and theother measures creatine plus creatinine. The sensors are made using acellulose acetate - glutaraldehyde method. Anal Chem 67, 2776, 1995teaches electropolymerization to immobilize the creatinine enzymes ontoan electrode. A poly(carbamoyl)sulphonate hydrogel is used in Anal ChimActa 325, 161, 1996. None of the above documents teaches the use ofmicrofabrication. Microdispensing to establish enzyme gel layers ontoelectrodes made by microfabrication is, however, disclosed in Anal ChimActa 319, 335, 1996.

[0014] Despite the recent and significant advances in analyte sensorsexemplified by U.S. Pat. Nos. 5,200,052 and 5,096,669, there remains aneed in the art for improved microfabrication techniques and greatercontrol of analyte flux. There is further a need in the art for reducingor eliminating the effect of interferant molecules on sensormeasurement.

[0015] The measurement of glucose with a microfabricated sensor,described in U.S. Pat. No. 5,200,051, assigned to i-STAT Corporation,uses a thin contiguous analyte attenuation (AA) layer made from asilicone copolymer to cover an enzyme layer. It provides a membrane thatis freely permeable to oxygen but is poorly permeable to glucose. Thisenables a linear response over the full range of glucose concentrationsfound in blood. As the '051 patent makes clear, oxygen is required instoichiometric amounts to sustain the enzymatic reaction, despite thelow levels generally present in blood. Using this membrane achieves thisgoal. The '051 patent includes a discussion of the general properties ofa microfabricated analyte attenuation layer at column 12, beginning atline 57, with a more detailed description beginning at column 38, line19. The etch process for the AA layer is discussed beginning at column58, line 5. Structures with open perimeters for measuring glucose areillustrated in FIGS. 7A & 8A of the '051 patent, however, glucosetransport occurs through a polysiloxane copolymer layer.

[0016] Wholly microfabricated sensors, that is, sensors which areuniformly mass-produced by thin-film techniques and micro-manufacturingmethods, had not demonstrated utility in a clinical setting prior to the'051 patent. The '051 patent showed that the degree of complexityinvolved with the mass production of commercially viable biosensors wasmuch more formidable than those persons of ordinary skill in the artonce perceived. Of major concern was the compatibility of inherentlyharsh physical and chemical processes associated with the then existingcommercial microfabrication manufacturing methods.

[0017] An article by Eleccion (Eleccion, M. Electronics 1986, Jun. 2,26-30) describes the then current state of the art with regard tomicrosensors and makes brief references to active areas of researchincluding the detection of specific ions, gases, and biologicalmaterials. Progress in the area of field effect transistors (FETs) isnoted and problems and limitations with present manufacturing methodsare discussed.

[0018] It is also important to note that in current clinical settingsmedical practitioners commonly request analyses of one or morecomponents of a complex biological fluid such as whole blood. Currently,such analyses require a certain amount of processing of whole blood,such as filtration and centrifugation, to avoid contamination of theinstruments or to simplify subsequent measurements. Frequently, bloodsamples are sent to a remote central laboratory where the analyses areperformed. Patients and physicians are thus deprived of valuableinformation, which, in most cases, is not available for hours, sometimesdays. Clearly, substantial advantages could be envisaged if analyses onundiluted samples could be carried out and if instruments or sensorswere available perform real-time measurements. This can now be achievedusing the point-of-care blood analysis system described in U.S. Pat. No.5,096,669 (assigned to i-STAT Corporation).

[0019] Despite the recent and significant advances in chemical sensortechnology as exemplified by U.S. Pat. Nos. 5,200,051 and 5,096,669,there remains a need in the art for improved microfabrication techniquesand greater control of analyte flux. There is further a need in the artfor reducing or eliminating the effect of interferant molecules onsensor measurement.

DISCLOSURE OF THE INVENTION

[0020] It is accordingly an aspect of the invention to provide achemical sensor capable of precise control of analyte diffusion rates orfluxes.

[0021] It is another aspect of the invention to provide a chemicalsensor having a layer with one or more apertures for diffusional fluxrate control and for controlling the stoichiometric ratio of co-reactantand analyte entering an enzyme layer.

[0022] It is another aspect of the invention to provide a sensor wherethe output characteristics thereof are not limited by the co-reactantconcentration in a sample, and where the response of the sensor isessentially linear over the entire range of analyte concentrationscommonly found in the sample.

[0023] It is another aspect of the invention to provide a chemicalsensor that is single use and storable in a dry state, but whichundergoes rapid wet-up on contacting a calibrant fluid.

[0024] It is yet another aspect of the invention to provide a chemicalsensor that can be manufactured with a high degree of consistency fromdevice to device in terms of both physical dimensions and outputcharacteristics.

[0025] Yet another aspect of the invention is to provide a chemicalsensor having a microfabricated diffusion layer or barrier of controlledgeometry and character, that is permeable to a selected analytemolecule, and is interposed between a microfabricated aperture and alayer containing a catalyst (optionally an enzyme) that can interactwith the analyte molecule.

[0026] It is yet another aspect of the invention to provide a chemicalsensor with a layer that specifically screens out interferant speciesbefore they reach the enzyme layer.

[0027] Another aspect of the invention is to provide a chemical sensorthat is single use and can be incorporated into a disposable cartridgefor testing blood samples at a bedside and remote locations.

[0028] It is yet another aspect of the invention to provide a chemicalsensor incorporating electrochemical optical and other sensingtechnologies that are amenable to substantially planar fabrication, forexample, an acoustic wave sensor.

[0029] It is yet another aspect of the invention to provide methods formanufacturing the above-described chemical sensor.

[0030] These aspects, and others set forth more fully hereinbelow, areachieved by a microfabricated device for detecting an analyte moleculein a liquid sample which also contains a co-reactant, e.g. oxygen, whichcomprises: a transducing element; a first layer having a first sidecontacting the surface of said transducing element, the first layercomprising a support matrix containing at least one enzyme capable ofcatalyzing the conversion of said analyte and co-reactant into areaction product detectable by the transducing element; a second layerin contact with the first layer, the second layer permitting transportof the analyte molecule and co-reactant; and a third layer covering thefirst and second layer, the third layer being permeable to co-reactantbut substantially impermeable to the analyte molecule and containing atleast one microfabricated aperture extending there through, whichpermits controlled transport of the analyte to the first layer.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] For a full understanding of the invention, the following detaileddescription should be read in conjunction with the drawings, wherein:

[0032]FIG. 1 is a schematic of one embodiment of the invention,illustrating a structure where the substrate (analyte molecule) can onlyenter the enzyme layer by passing through an edge-plane diffusion layer,whereas oxygen (co-reactant) passes in through a gas permeable layer.

[0033]FIG. 2 is a schematic illustrating another embodiment wherein thesubstrate can only enter the enzyme layer by passing through anedge-plane interferant removal layer, whereas oxygen passes in throughthe gas permeable layer.

[0034]FIG. 3 is a schematic illustrating another embodiment wherein thesubstrate can only enter the enzyme layer by passing through theedge-plane interferant removal and diffusion layers, whereas oxygenpasses in through the gas permeable layer.

[0035]FIG. 4 is a schematic illustrating an embodiment of the inventionwherein the substrate can only enter the enzyme layer by passing throughthe edge-plane interferant removal and diffusion layers, whereas oxygenpasses in through the gas permeable layer. In this embodiment theinterferant removal layer extends beyond the gas permeable layer.

[0036]FIG. 5 is a schematic illustrating another embodiment wherein thesubstrate can only enter the enzyme layer by passing through a pinholeor slot-shaped opening in the gas permeable layer and diffusion layer,whereas oxygen passes in through the gas permeable layer.

[0037]FIG. 6 is a schematic illustrating another embodiment wherein thesubstrate can only enter the enzyme layer by passing through a pinholeor slot-shaped opening in the gas permeable layer and the interferantremoval layer whereas oxygen passes in through the gas permeable layer.

[0038]FIG. 7 is a schematic illustrating another embodiment wherein thesubstrate can only enter the enzyme layer by passing through a pinholeor slot-shaped opening in the gas permeable layer, interferant removallayer and diffusion layer, whereas oxygen passes in through the gaspermeable layer.

[0039]FIG. 8 is a schematic illustrating another embodiment wherein thesubstrate can only enter the enzyme layer by passing through a pinholeor slot-shaped opening in the gas permeable layer and the diffusionlayer, whereas oxygen passes in through the gas permeable layer.

[0040]FIG. 9 is a graph showing the response to both creatine andcreatinine of the embodiment of FIG. 3, where the interferant screeninglayer is designed to remove creatine. The response to equimolarcreatinine and creatine concentrations over the physiological range isessentially linear and shows that about 90% of the creatine is screened.Note that increasing the length and enzyme loading in the screeninglayer can improve further upon the screening of creatine, thusincreasing the specificity of the device to creatinine.

[0041]FIG. 10 is a graph showing a comparison for the embodiment of FIG.3 against a non-microfabricated commercially available creatinine assayon actual blood samples taken from patients. These data show that thenew device gives equivalent results and can therefore be usedclinically. Data were obtained in accordance with the teaching of U.S.Pat. No. 5,112,455.

[0042]FIG. 11 is a graph showing the response of a lactate sensor of theinvention.

[0043]FIG. 12 is a graph of the correlation of the lactate sensor with alactate assay in whole blood samples.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] The present invention relates to wholly microfabricated chemicalsensors useful in measuring constituents (analytes) in various fluids.While the bulk of following detailed description concerns the use ofchemical sensors to measure analytes found in biological fluids such asblood, it is to be understood that the invention encompasses use of thesensors in non-biological applications as well. Likewise, the term“analyte” is to be construed broadly as encompassing both ionic andnon-ionic species or molecules contained or dissolved in a fluid,including dispersions. The terms “chemical sensors” and “biosensors” areused interchangeably hereinafter.

[0045] The microfabrication processes of the invention establish aplurality of thin films and related structures over a planar wafer in afashion which allows reproducibility and control over the dimensionalfeatures of the overlaid structures. In the present invention, suchreproducibility and dimensional control have been realized at the waferlevel for the mass production of chemical sensors, which sensorsincorporate biologically active macromolecules and other reagentsnecessary for the conversion of selected analyte molecules to morereadily detectable species.

[0046] This invention also relates to novel electrochemical assayprocedures and to novel wholly microfabricated biosensors useful indetermining the presence and/or concentration of biological species(analytes) of interest. The invention also relates to a substrate oranalyte that does not undergo direct detectable electrochemicaloxidation or reduction but which undergoes a reaction with a substrateconverter, generally an enzyme, that produces changes in theconcentration of electroactive or an optically detectable species. Thesechanges are measured and related proportionately to the concentration ofthe analyte of interest. Additionally, the invention pertains to methodsfor making the sensor.

[0047] The wholly microfabricated chemical sensor of the presentinvention comprises multiple elements. The following is a generaldescription of the process for forming the chemical sensor of theinvention.

[0048] The transducing element is formed on a substantially planarsurface, generally a silicon wafer or an optically transparent material.For biosensors based on optical detection the transducing element may bethe optically transparent surface onto which other layers are added.Means for supplying excitation wavelengths and adapting opticaldetectors to such surfaces are well known in the art. For biosensorsbased on electrochemical detection, e.g. amperometic, potentiometric andconductimetric, means for microfabricating these base sensors ortransducing elements onto a planar surface are disclosed in U.S. Pat.No. 5,200,051, which is incorporated herein by reference in itsentirety. Additional structures are then established over the resultingtransducing element, which additional structures may include asemipermeable solid film or permselective layer capable of acting as abarrier against interfering chemical species while allowing thetransport of smaller detectable chemical moieties of interest. Thesedetectable chemical moieties are typically electroactive molecules andmay include low molecular weight ionic species, oxygen, hydrogenperoxide and small redox mediator molecules known in the art.Alternatively, the detectable chemical moieties may be dyes or otheroptically detectable species generally used in enzyme assays and wellknown in the art.

[0049] The semipermeable solid film may further comprise materials,compounds or molecules that may serve to sensitize the base sensor to apreselected ionic species (e.g., ammonium ion). Most noteworthy are thesupport matrices described in the instant invention which matricespossess the physical and chemical features necessary to support thevarious bioactive molecules that constitute the principal means forconverting the particular analytes in a given analytical sample intodetectable and/or quantitatively measurable species at the transducingelement. Techniques are disclosed for localizing or patterning saidmatrices on certain desired areas of the wholly microfabricatedbiosensor which allow for the optimum control over dimensional featuresof the biolayers as well as the versatility to accommodate a wide rangeof bioactive molecules.

[0050] Additionally, the present invention also discloses materialswhich serve, in particular embodiments of the instant biosensor, asoverlaid structures which function as a barrier to the transport ofselected analyte species, which are present in high concentrations inthe sample. Such analyte barrier (AB) layers allow for a linear sensorresponse over a wide range of analyte concentrations via the presence ofapertures of defined dimensions and positioned at specific locations,which permit a controlled diffusional flux of analyte. Furthermore, theoverlaid AB layer, which is preferably derived from asiloxane-nonsiloxane copolymer, is capable of excluding very largemolecules or other contaminating constituents of the sample whose directcontact with the underlying structures would result in interference orfouling and an eventual reduction in the reliability of the biosensor.Suitable materials for forming the AB layer are described in U.S. Pat.No. 5,200,051, including the various siloxane-nonsiloxane copolymers setforth therein. In addition to these copolymers, there may be usedvarious polyurethanes, cellulose acetate, tetrafuoroethylene polymers,organic negative photoresists, organic positive photoresists, polyimidesand photoformable polyimides.

[0051] If the AB layer is of the appropriate structure and composition,it may also function as a gas permeable membrane. In certain embodimentsof the present invention, such a gas permeable membrane has thepractical advantage of allowing only very small molecules to passthrough. These molecules can act as co-reactants in reactions whereanalyte molecules are converted to species that are detectable at thetransducing element.

[0052] The AB layer of the instant invention is established on thesubstrate wafer or any intervening structures with the kind ofdimensional, localized, and geometric control which is compatible withother steps in the overall microfabrication process of the instantinvention and the notion of an automated, wafer-level mass production ofbiosensors.

[0053] Quite apart from the AB layer mentioned above, a semipermeablesolid film which is able to function as a molecular weight-sensitivetransmissive film is among the layers which can be established by themethods of the present invention. Depending upon the composition andfinal thickness of this semipermeable solid film, also referred to as apermselective layer, molecules having molecular weights above a giventhreshold can be effectively excluded from entering and diffusingthrough such a film. As a general illustration of the function andutility of this permselective layer, molecules having a molecular weightof about 120 or above are effectively blocked by a solid film having athickness of about 5 to about 10 nm. Varying degrees of control over thesize of the molecules excluded and the rates of transport of smallermolecules which are able to diffuse through the solid film can beobtained with solid films having a thickness in the range of about 2 toabout 50 nm. With certain types of materials, these permselective layersmay be as thin as 1 nm or may be as thick as 100 nm.

[0054] In a preferred embodiment of an amperometric glucose biosensor, alayer of iridium is sputtered onto a silicon wafer and then patternedusing established microfabrication processes to form an electrode(diameter 200 μm) as the transducing element. A permselective layer thatpermits transport of hydrogen peroxide is then patterned over theiridium electrode according to U.S. Pat. No. 5,212,050, which isincorporated herein by reference in its entirety. A mixture ofdichromated photoformable gelatin and the enzyme glucose oxidase is thenspin coated onto the wafer and patterned to form a layer of thickness ofabout 1.0 μm directly over the electrode, according to the teaching ofU.S. Pat. No. 5,200,051. This is followed by patterning a second gelatindiffusion channel (or layer of thickness about 1.0 μm) that partiallycovers the enzyme layer and extends beyond the perimeter of the enzymelayer by 50 μm. A thick AB layer formed from a siloxane-nonsiloxanecopolymer is then spin-coated onto the wafer and patterned according tothe method disclosed in U.S. Pat. No. 5,200,051 to establish a layerthat encloses the first two layers.

[0055] Unlike embodiments described in the '051 patent, however, the ABlayer thickness (for example, about 1.0 μm) is sufficient to eliminatedetectable glucose permeation directly through this layer. However, theAB layer is still freely permeable to oxygen. A cap layer made fromdichromated gelatin is then established in the same way as described inthe '051 patent, except that a novel mask design is used to provide forapertures to be formed at specific locations in the AB layer. When theAB layer is etched in the standard manner disclosed in the '051 patent,small apertures (e.g., 5 μm diameter) are made in the AB layer in theregion above the diffusion barrier layer (but not in the region directlyabove the enzyme layer) through which glucose can pass. Preferably, thesize of the aperture(s) is at least about 0.01 μm by 1.0 μm(rectangular) or, if circular, having a diameter of from about 0.5 μm toabout 100 μm. Desirably the diameter is from about 2 μm to about 10 μm.Rectangular apertures can be from about 1 μm to about 20 μm on the shortside and from about 10 μm to about 3000 μm on the long side. Desirably,the dimensions of rectangular apertures are from about 3 μm to about 12μm on a short side and from about 50 μm to about 2000 μm on a long side.In a preferred embodiment, the rectangular apertures have dimensions ofabout 5 μm×1000 μm.

[0056] The apertures may also form an annulus, the thickness of whichcan vary similar to the diameter dimensions given above.

[0057] In certain circumstances it is desirable that a portion of thediffusion layer also contains one or more enzymes that can eliminatespecific interferant molecules, e.g. ascorbate and urate. For examplethe diffusion layer can incorporated ascorbate oxidase or uricase or thelike.

[0058] The biosensor of the invention may be operated amperometricallyin conjunction with a silver-silver chloride reference electrode whichis external to the device or incorporated adjacent to the iridiumelectrode as disclosed in U.S. Pat. No. 5,200,051. Means for activatingmicrofabricated sensors and obtaining reliable data in aqueous andwhole-blood samples are disclosed in U.S. Pat. No. 5,112,455 which isincorporated herein by reference in its entirety. Generally, thisinvolves comparing the response of the sensor with a reference electrodein both a calibrant fluid and a sample fluid, relating the signalmeasurements and then determining the concentration of analyte speciesin the sample fluid based on the signal relationship. The devicedescribed here gives reliable glucose measurements in venous wholeblood, which generally has a very low oxygen (co-reactant) concentration(ca. 50 μm) over the entire range of glucose (analyte molecule)concentrations found in samples from diabetics (ca. 1-30 mM).

[0059] With these new devices, it is practical for the aperture(s) to bemicron-sized in only one or in both of the x-y dimensions. Thus, forexample, an aperture can be a 5 μm circular pore, a slot of dimensions 5μm×1000 μm, or a ring 5 μm wide and has a perimeter of 1000 μm. Inaddition, the aperture(s) can be positioned directly above thetransducing element, or adjacent to the transducing element or formed bypatterning the AB layer to leave an exposed edge at the outer perimeterof the diffusion layer.

[0060] Another alternative is to make the aperture in the z-x or z-ydimension. This can be achieved by patterning the AB layer to leave anexposed perimeter edge of the second layer. This enables the height(thickness, z-dimension) of the second layer to control one dimension ofthe aperture while the length of the exposed perimeter controls theother dimension. As the second layer can be spin coated on to a waferwith a controlled thickness in the range 0.01 μm to 2 mm (which is wellknown in the microfabrication art), using this method it is possible tofabricate essentially rectangular apertures in the z-x and z-y planes assmall as 0.01 μm z-dimension and 1.0 μm x-dimension. This providesadditional means for controlling the flux of an analyte molecule to thecatalyst layer. The particular dimensions of the aperture(s) can bedetermined by the skilled artisan and are a function of, inter alia, theanalyte flux desired and the stoichiometry of the reaction

[0061] Accurate dimensional control at these geometries is onlyattainable using microfabrication. Negative and positive organicphotoresists, polyurethane, cellulose acetate, polyimide andphotoformable polyimide and the like can be used instead of asiloxane-nonsiloxane copolymer to act as co-reactant-permeable,analyte-impermeable AB layers. Where these materials are photoformable,an aperture can be formed directly by exposure and development.Otherwise a patterning method as for the siloxane-nonsiloxane copolymeris required as described above.

[0062] In addition to dichromated gelatin and gelatin containing ferricchloride and other photoactivators as described in U.S. Pat. No.5,200,051, it is also possible to use various other hydrogel materials,for example, a photoformable polyvinyl alcohol material to form both theenzyme layer and the diffusion (or interferant screening) layer. Forcertain enzymes, e.g. creatinase, the latter enables retention of higherlevels of activity.

[0063] The general theory applicable to biosensors is well known in theart. For example, U.S. Pat. No. 4,484,987 discloses an equation relatingbulk concentrations to those in the enzyme layer. Unlike the '987patent, embodiments of the present invention include an analytediffusion layer. If a diffusion coefficient, D, for a typical analytemolecule is 10⁻⁶ cm⁻²s⁻¹ in a gelatin or polyvinyl alcohol layer, thediffusion length, l, can be estimated from l= (2Dt)^(½), which impliesthat these molecules can diffuse about 10 μm in the first second in theplane of the layer. Note that the diffusion coefficient can becontrolled by the degree of crosslinking in the layer, e.g. more or lessphoto-initiator or photo-crosslinker in the matrix, or subsequenttreatment with glutaraldehyde or another crosslinker. A method forincreasing the porosity and increasing the diffusion coefficient is toadd albumin or another globular protein to the matrix before patterning.Thus by changing the length, thickness and composition of the diffusionlayer, it is possible to control the response time and the degree thedevice exhibits an extended linear output response to the analytemolecule even at low co-reactant levels. For example, it has been foundthat a planar 10-200 μm diffusion layer between the aperture and theenzyme layer over the transducing element may be used advantageously. Itis only by using microfabrication processes, unlike the approachesapplied by the prior art, that attaining accurate control and a highlevel of device to device reproducibility necessary for commercialutility, is possible.

[0064] Several embodiments of the invention are shown in the Figures.FIG. 1 illustrates a first embodiment wherein a biosensor is indicatedgenerally by the number 10. A transducing element 12 is placed over aplanar surface 14 and an enzyme layer 16 is positioned over thetransducing element 12. Surrounding the enzyme layer 16 and thetransducing element 12 is a diffusion layer 18 having an edge surface20. Placed over the enzyme layer 16 and the diffusion layer 18 is ananalyte barrier layer 22. A cap 24, used in photoforming apertures inthe analyte barrier layer 22, is located over the layer 22. Analyte,such as glucose or creatinine, as well as oxygen, diffuses through thediffusion layer 18 through the edge surface 20. The analyte barrierlayer 22, however, permits only oxygen (and other molecules of similarsize) to pass through, while preventing larger molecules includinganalyte, from passing. Thus oxygen diffusion to the enzyme layer 16occurs over a much larger surface area than does analyte diffusion,thereby compensating for the lower oxygen concentration in a bloodsample compared to analyte concentration. Oxygen and analyte are thus atsubstantially stoichiometric concentration at the enzyme layer 16. TheAB layer 22 contains one or more apertures (not shown) which expose thesurface of the enzyme layer 15 and/or the diffusion layer 18 to analyte.

[0065] A modification of the biosensor of FIG. 1 is illustrated in FIG.2. In this embodiment the diffusion layer 18 of FIG. 11 is replaced byan interferant removal layer 30 which contains one or more enzymes orcatalysts which react with molecules having a potential for interferingwith the analysis. The interferant removal layer 30 may be constructedof the same materials as that of the diffusion layer 18, thus providingthe dual function of a diffusion layer and interferant removal.

[0066] In a preferred embodiment, the various elements of the sensor ofFIG. 2 comprise a sensor including a noble metal electrode whichfunctions as an electrocatalyst for H₂O₂ electrooxidation; a gammaaminosilane layer which functions to prevent redox-active species thatare larger than hydrogen peroxide, e.g. ascorbate and urate, fromreaching the electrode surface; an enzyme layer to convertnon-electrochemical reactive analyte molecules to hydrogen peroxide; adiffusion layer, and an AB layer from about 0.1-2 μm thick thateliminates analyte molecules such as glucose and creatinine permeatingdirectly into the enzyme layer, but is still freely permeable to oxygenand water. A cap layer is established in the same way as the standardglucose process but the mask provides for apertures to be formed atspecific locations. When the AB layer is etched in the standard manner,apertures are made in the AB layer through which analyte molecules canpass. Thus, in this design, the substrate diffusion into the enzymelayer is regulated by the number and size of the apertures in the ABlayer and the length of the diffusion path.

[0067]FIG. 3 illustrates an embodiment similar to FIGS. 1 and 2 exceptthat it includes both a diffusion layer 40 and an interferant removallayer 42.

[0068] The embodiment of FIG. 4 is similar to that of FIG. 3 in that itincludes separate diffusion and interferant removal layers. However, inthis embodiment, the interferant removal layer 50 extends over the edgesof the AB layer 52 and cap 54 and covers a portion of the upper surface56 of cap 54.

[0069] In the embodiment of FIG. 5, the AB layer 60 covers the enzymelayer 62. A pinhole 64 is provided through the cap 66 and AB layer 60 toallow substrate to contact the diffusion layer 68. In this embodiment,the electrode 66 and enzyme layer 62 are positioned outward from thediffusion layer 68.

[0070]FIG. 6 illustrates an embodiment similar to FIG. 5 except that thediffusion layer is replaced by an interferant removal layer 70.

[0071]FIG. 7 illustrates an embodiment configured similar to FIGS. 5 and6 and incorporating both an interferant removal layer 80 and a diffusionlayer 82.

[0072]FIG. 8 provides for an interferant removal layer 90 positionedover the cap 92 and having an AB layer 94 covering the edge portion ofthe enzyme layer 96.

[0073] The process parameters expand on those disclosed in U.S. Pat. No.5,200,051. Both platinum and iridium electrodes (diameter 200-360 μm)are used with the standard gamma aminosilane processes in U.S. Pat. No.5,212,050. Glucose oxidase is immobilized in a dichromated gelatinlayer, thickness 0.1-2.0 μm. The standard AB etch time was optimized toensure no under or over-etch of the aperture, i.e. provide for precisecontrol of aperture diameter.

[0074] The determination of creatinine is a good example of an analytethat requires a screening layer to remove interferants. The measurementof creatinine utilizes three enzymes to convert creatinine to hydrogenperoxide. These enzymes are CNH (creatinine a amidohydrolase also calledcreatininase), CRH (creatine amidinohydrolase also called creatinase)and SAO (sarcosine oxidase) which catalyze the following reactions,respectively.

[0075] creatinine→creatine+H₂O

[0076] creatine→sarcosine+urea

[0077] sarcosine+oxygen→glycine+formaldehyde+H₂O₂

[0078] The reaction is complicated by the fact that blood naturallycontains both creatinine and creatine, thus it is necessary to removethe endogenous creatine before creatinine can be accurately measured.Those skilled in the art will understand that any creatinine thatreaches the enzyme layer would produce an erroneous background signal inthe determination of creatinine. This problem is solved by creating acreatine-screening layer as part of the creatinine sensor.

[0079] For example, the device shown in FIG. 3 is a creatinine sensorthat has a diffusion layer and interferant removal layer for creatine.Its function is to prevent the diffusion of the endogenous creatine tothe enzyme layer by converting it to non-interfering substances. Thislayer contains the enzymes CRH, SAO and catalase (CAT). The latterconverts hydrogen peroxide to water and oxygen, thus preventing thehydrogen peroxide from diffusing to the transducing element. Note thatthis combination of enzymes permits the creatinine to diffuse throughthe screening layer without reaction, and thus reach the enzyme layer.

[0080] In this example, enzymes in both layers are immobilized in aphotoformable polyvinyl alcohol bearing styrylpyridinium groups(PVA-SbQ). The enzyme layer is confined within the perimeter of theunderlying platinum electrode, whereas the diffusion layer and theinterferant removal layer extends beyond (20 to 50 μm) the perimeter ofthe electrode. An AB layer (about 1-2 μm) is then spin-coated over theselayers. The thickness of the AB is sufficient to eliminate creatinine(and creatine) permeation into the enzyme layer, but still be freelypermeable to oxygen and water. The AB layer is then patterned so that itdoes not completely enclose the screening layer, thus providing adiffusion path for creatinine (and creatine). The complete creatininemicrofabrication process is described in the attached table.

[0081] In another embodiment, where reagents other than an enzyme, e.g.ATP, glycerol, a redox mediator molecule, an organic dye molecule, arerequired in the enzyme or screening layers for reliable sensoroperation, these materials may be introduced as part of the enzyme orscreening layer matrix deposition process, impregnated after the layershave been established but prior to deposition of the AB layer, adsorbedthrough the microfabricated apertures after they are formed, or evenadsorbed through the apertures as part of the calibration process priorto contacting the sensor with the sample.

[0082] The conversion of lactate is catalyzed by the enzyme lactateoxidase and produces hydrogen peroxide, which is detected at a platinumelectrode.

[0083] In a preferred embodiment, the platinum electrode (360 μmdiameter) is coated with a gamma aminosilane layer (described above)over which is patterned an enzyme layer (360 μm diameter, polyvinylalcohol bearing styrylpyridinium groups and lactate oxidase). Adiffusion layer (diameter 560 μm, thickness 1.0 μm) is patterned overthe enzyme layer and extends beyond its perimeter. Asiloxane-nonsiloxane layer is patterned to enclose the entire structure,except for a concentric annular aperture (width 40 μm) which permitslactate to enter the diffusion layer 40 μm beyond the perimeter of theenzyme layer. FIG. 11 shows that the response of the sensor isessentially linear in aqueous samples that correspond to the range oflactate concentrations found in physiological samples. FIG. 12 showsthat the sensor provides results that correlate with a commerciallyestablished lactate assay in whole-blood samples.

[0084] Other related embodiments, based on the disclosure will beapparent to those skilled in the art.

What is claimed is:
 1. A microfabricated device for detecting an analytemolecule in a co-reactant-containing sample comprising (a) a transducingelement; (b) a first layer contacting the surface of said transducingelement, said first layer comprising a support matrix containing atleast one catalyst capable of catalyzing the conversion of said analyteand co-reactant into a reaction product detectable by said transducingelement; (c) a second layer in contact with said first layer, secondlayer permitting transport of said analyte molecule and co-reactant; and(d) a third layer covering said first and second layers, said thirdlayer being permeable to co-reactant but substantially impermeable tosaid analyte molecule and containing at least one microfabricatedaperture extending there through, which permits transport of saidanalyte to said first layer.
 2. A microfabricated device as claimed inclaim 1 , wherein the second layer extends beyond the perimeter of thefirst layer.
 3. A microfabricated device as claimed in claim 1 , whereinthe at least one aperture in said third layer extends to the firstlayer.
 4. A microfabricated device as claimed in claim 1 , where theaperture is in the plane of the third layer.
 5. A microfabricated deviceas claimed in claim 1 , where the aperture is in the perimeter of thesecond layer.
 6. A microfabricated device as claimed in claim 5 , wherethe aperture in the second layer is at least about 0.01 μm by 1.0 μm. 7.A microfabricated device as claimed in claim 1 , wherein the at leastone aperture in said third layer extends to a surface of the secondlayer.
 8. A microfabricated device as claimed in claim 1 , comprising aplurality of apertures in said third layer.
 9. A microfabricated deviceas claimed in claim 8 , wherein the plurality of apertures aresubstantially circular.
 10. A microfabricated device as claimed in claim1 , wherein the diameter of the aperture is from about 0.5 μm to about100 μm.
 11. A microfabricated device as claimed in claim 1 , wherein thediameter of said aperture is from about 2 μm to about 10 μm.
 12. Amicrofabricated device as claimed in claim 1 , wherein the diameter ofsaid aperture is about 5 μm.
 13. A microfabricated device as claimed inclaim 1 , wherein the aperture is rectangular.
 14. A microfabricateddevice as claimed in claim 13 , wherein the aperture has dimensions offrom about 1 μm to about 20 μm on a short side and from about 10 μm toabout 3000 μm on a long side.
 15. A microfabricated device as claimed inclaim 13 , wherein the aperture has dimensions of from about 3 μm toabout 12 μm on a short side and from about 50 μm to about 2000 μm on along side.
 16. A microfabricated device as claimed in claim 13 , whereinthe aperture has dimensions of about 5 μm on a short side and about 1000μm on a long side.
 17. A microfabricated device as claimed in claim 1 ,wherein the plurality of apertures are substantially annular.
 18. Amicrofabricated device as claimed in claim 7 , comprising a plurality ofapertures extending to a surface of the second layer.
 19. Amicrofabricated device as claimed in claim 1 , wherein the first layercomprises a photoformable material where the matrix component isselected from the group comprising a proteinaceous material, a gelatin,a hydrogel, a hydrophilic organic polymer and polyvinyl alcohol, and thephotoactive material is selected from the group comprising dichromate,ferric chloride a styrylpyridinium salt and a stilbizonium salt.
 20. Amicrofabricated device as claimed in claim 1 , wherein the second layercomprises a photoformable material where the matrix component isselected from the group comprising a proteinaceous material, a gelatin,a hydrogel, a hydrophilic organic polymer and polyvinyl alcohol, and thephotoactive material is selected from the group comprising dichromate,ferric chloride, a styrylpyridinium salt and a stilbizonium salt.
 21. Amicrofabricated device as claimed in claim 1 wherein the catalyst is anenzyme or combination of enzymes.
 22. A microfabricated device asclaimed in claim 21 , wherein the enzyme or combination of enzymes isselected from the group comprising glucose oxidase, lactate oxidase,pyruvate oxidase, cholesterol oxidase, bilirubin oxidase, sarcosineoxidase, creatinase and creatininase cholesterol esterase.
 23. Amicrofabricated device as claimed in claim 1 , wherein the second layercomprises a photoformable gelatin or polyvinyl alcohol layer.
 24. Amicrofabricated device as claimed in claim 1 , wherein the third layeris selected from the group comprising a silicone copolymer,polyurethane, cellulose acatate, a siloxane-nonsiloxane copolymer, atetrafluoroethylene polymer, an organic negative photoresist, an organicpositive photoresist, polyimide or a photoformable polyimide.
 25. Amicrofabricated device as claimed in claim 1 , wherein the co-reactantis oxygen.
 26. A microfabricated device as claimed in claim 1 , whereinthe analyte molecule is selected from the group comprising glucose,creatine, cholesterol, lactate, pyruvate, sarcosine or bilirubin.
 27. Amicrofabricated device as claimed in claim 1 , wherein the first layerhas a thickness of from about 0.01 μm to about 2 mm, the second layerhas a thickness of from about 0.01 μm to about 2 mm, and the third layerhas a thickness of from about 0.01 μm to about 2 mm.
 28. Amicrofabricated device as claimed in claim 1 , wherein the catalystreaction produces an electrochemically detectable reaction product. 29.A microfabricated device as claimed in claim 28 , wherein theelectrochemically detectable reaction product is selected from the groupcomprising, oxygen, hydrogen peroxide, a redox mediator, carbon dioxide,hydrogen ion, potassium ion, sodium ion, ammonium ion, calcium ion,fluoride ion.
 30. A microfabricated device as claimed in claim 1 whereinthe transducer element is an amperometric, potentiometric orconductimetric electrode.
 31. A microfabricated device as claimed inclaim 1 , where the catalyst reaction produces an optically detectablereaction product.
 32. A microfabricated device as claimed in claim 1 ,wherein said transducer element is an optical detector.
 33. Amicrofabricated device as claimed in claim 1 , wherein the second layercompletely covers, partially covers or abuts the edge of the firstlayer.
 34. A microfabricated device as claimed in claim 1 , wherein thesecond layer incorporates one or more reagent for converting one or moreinterferant species to non-interferant species.
 35. A microfabricateddevice as claimed in claim 1 , wherein a portion of the second layerthat is not in direct contact with the first layer incorporates one ormore reagent for converting one or more interferant species tonon-interferant species.
 36. A microfabricated device as claimed inclaim 34 , wherein one or more reagents are selected from the groupascorbate oxidase, uricase, sarcosine oxidase, creatinase, catalase,biliribin oxidase, lactate oxidase, pyruvate oxidase and glucoseoxidase.
 37. A process for manufacturing a planar microfabricated devicefor detecting an analyte molecule in an oxygen-containing liquid samplecomprising: microfabricating a transducing element on a planar surface;microfabricating a first layer above said transducing element comprisingan enzyme and support matrix, said enzyme capable of converting saidanalyte and oxygen in a manner detectable at said transducing element;microfabricating a second layer above said first layer that is permeableto both the analyte molecule and oxygen; establishing a third layerabove said first layer comprising a polymer that is permeable to oxygen,but impermeable to said analyte; establishing a photoformable layer oversaid third layer; exposing said photoformable layer through a mask, saidmask containing a pattern for forming one or more apertures ofcontrolled geometry at predetermined locations in said photoformablelayer; developing said pattern; contacting said photoformed layer withan etchant capable of etching through said third layer to produce athird layer containing one or more microfabricated apertures ofcontrolled geometry at predetermined locations capable of permittingtransport of said analyte to said first layer.
 38. A process as claimedin claim 37 , wherein the first layer comprises a photoformable materialwhere the matrix component is selected from the group comprising aproteinaceous material, a gelatin, a hydrogel, a hydrophilic organicpolymer and polyvinyl alcohol, and the photoactive material is selectedfrom the group comprising dichromate, ferric chloride a styrylpyridiniumsalt and a stilbizonium salt.
 39. A process as claimed in claim 37 ,wherein the second layer comprises a photoformable material where thematrix component is selected from the group comprising a proteinaceousmaterial, a gelatin, a hydrogel, a hydrophilic organic polymer andpolyvinyl alcohol, and the photoactive material is selected from thegroup comprising dichromate, ferric chloride a styrylpyridinium salt anda stilbizonium salt.
 40. A process as claimed in claim 37 , wherein theenzyme or combination of enzymes is selected from the group comprisingglucose oxidase, lactate oxidase, pyruvate oxidase, cholesterol oxidase,bilirubin oxidase, sarcosine oxidase, creatinase and creatininasecholesterol esterase.
 41. A microfabricated device as claimed in claim37 , wherein the third layer is selected from the group comprising asilicone copolymer, polyurethane, cellulose acatate, asiloxane-nonsiloxane copolymer, a tetrafluoroethylene polymer, anorganic negative photoresist, an organic positive photoresist, polyimideor a photoformable polyimide.
 42. A process as claimed in claim 37 ,wherein the analyte molecule is selected from the group comprisingglucose, creatine, cholesterol, lactate, pyruvate, sarcosine orbilirubin.
 43. A process for manufacturing a planar microfabricateddevice for detecting an analyte molecule in an oxygen-containing liquidsample comprising: microfabricating a transducing element on a planarsurface; microfabricating a first layer above said transducing elementcomprising an enzyme and support matrix, said enzyme capable ofconverting said analyte and oxygen in a manner detectable at saidtransducing element; microfabricating a second layer above said firstlayer that is permeable to both the analyte molecule and oxygen;establishing a third layer which is photoformable above said first layerthat is permeable to oxygen, but impermeable to said analyte; exposingsaid photoformable layer through a mask, said mask containing a patternfor forming one or more apertures of controlled geometry atpredetermined locations; developing said pattern to produce a thirdlayer containing one or more microfabricated apertures of controlledgeometry at predetermined locations capable of permitting transport ofsaid analyte to said first layer.
 44. A process as claimed in claim 43 ,wherein the first layer comprises a photoformable material where thematrix component is selected from the group comprising a proteinaceousmaterial, a gelatin, a hydrogel, a hydrophilic organic polymer andpolyvinyl alcohol, and the photoactive material is selected from thegroup comprising dichromate, ferric chloride a styrylpyridinium salt anda stilbizonium salt.
 45. A process as claimed in claim 43 , wherein thesecond layer comprises a photoformable material where the matrixcomponent is selected from the group comprising a proteinaceousmaterial, a gelatin, a hydrogel, a hydrophilic organic polymer andpolyvinyl alcohol, and the photoactive material is selected from thegroup comprising dichromate, ferric chloride a styrylpyridinium salt anda stilbizonium salt.
 46. A process as claimed in claim 43 , wherein theenzyme or combination of enzymes is selected from the group comprisingglucose oxidase, lactate oxidase, pyruvate oxidase, cholesterol oxidase,bilirubin oxidase, sarcosine oxidase, creatinase and creatininasecholesterol esterase.
 47. A microfabricated device as claimed in claim43 , wherein the third layer is selected from the group comprising asilicone copolymer, polyurethane, cellulose acatate, asiloxane-nonsiloxane copolymer, a tetrafluoroethylene polymer, anorganic negative photoresist, an organic positive photoresist, polyimideor a photoformable polyimide.
 48. A process as claimed in claim 43 ,wherein the analyte molecule is selected from the group comprisingglucose, creatine, cholesterol, lactate, pyruvate, sarcosine orbilirubin.
 49. A microfabricated device as claimed in claim 35 , whereinone or more reagents are selected from the group ascorbate oxidase,uricase, sarcosine oxidase, creatinase, catalase, biliribin oxidase,lactate oxidase, pyruvate oxidase and glucose oxidase.